The present invention relates to a novel and useful panel of monoclonal antibodies which may be employed in immunoassays and other procedures for detection and/or quantitation of human iNOS.
Nitric oxide (NO) has recently been recognized as an effector and/or regulator molecule. For example, a recent field of investigation focused on the activity of NO upon the activation of soluble guanylate cyclase, which is responsible for endothelial dependent relaxation in the vasculature. An article entitled xe2x80x9cImmunohistochemical Demonstration of a Paracrine Role of Nitric Oxide in Bronchial Functionxe2x80x9d by Rangassmy et al., er gan Physiological Society (1994) recognizes this effect with respect to bronchial blood vessels.
Concurrently, investigators have discovered that NO acts as a new neurotransmitter in the central and peripheral nervous system. In addition, activated macrophage cytotoxicity was found to be activated in host defense mechanisms based on the presence of NO. NO is now considered the smallest biosynthetically derived effector molecule secreted in mammalian systems. Reference is made to an article entitled xe2x80x9cThe Molecule of the Yearxe2x80x9d, Science Magazine, Volume 258 (December 1992), by Koshland, which elaborates on the physiological importance of NO.
An article entitled xe2x80x9cIncreased Production of Nitric Oxide By Neutrophils and Monocytes From Cirrhotic Patients With Ascites and Hyperdynamic Circulationxe2x80x9d, by Laffi et. al., Hepatology, Volume 22, No. 6, (1995) and an article entitled xe2x80x9cMolecular Cloning and Expression of Inducible Nitric Oxide Synthase from Human Hepatocytesxe2x80x9d by Geller et al., Proc. Natl. Acad. Sci. USA, Volume 90 (April 1993) describes activity of nitric oxide synthase (NOS) and of nitric oxide in the liver. The latter reference includes an amino acid sequence describing human inducible NOS. In general, these articles associate cirrhosis with its concomitant activation of hepatocytes due to the inflammation and destruction of the liver, with the induction of iNOS and the subsequent overproduction of NO.
Rejection of transplanted organs is proposed to be mediated by host defense mechanisms in which activated monocytes, macrophages, and/or neutrophils are active, and through the actions of iNOS leads to the inevitable production of NO. Others have attempted to develop drugs which specifically inhibit iNOS, thus stopping the production of NO, without simultaneously inhibiting either neuronal NOS (nNOS) or endothelial (eNOS), the other two isofroms of this enzyme.
An article entitled xe2x80x9cIncreased Nitric Oxide Synthase Activity Despite Lack of Response to Endothelium-dependent Vasodilators in Postischemic Acute Renal Failure in Ratsxe2x80x9d, by Conger et al., The Journal of Clinical Investigations, Inc., Volume 96 (July 1995) recognizes nitric oxide activity in the failure of rat kidneys.
An article entitled xe2x80x9cImmunohistochemistry in the Identification of Nitric Oxide Synthase Isoenzymes in Myocardial Infarctionxe2x80x9d, by Wildhirt et al., Cardiovascular Research, Volume 29 (1995) recognizes the conversion of L-arginine to citrulline and nitric oxide in infarcted rabbit myocardium, which leads to damage of the heart.
The NO biosynthetic pathway has been extensively examined recently. It is now recognized that there is a family of isozymes which produce NO. An article entitled xe2x80x9cThe Nitric Oxide Synthase Family of Proteinsxe2x80x9d, by Sessa, J. Vasc. Res. (1994) recognizes the trio of NOS isozymes. All three NOS isozymes catalyze the conversion of L-arginine and oxygen to citrulline and NO. In addition, five co-factors have also been found to be required for this catalytic conversion. These are calmodulin, NADPH, FAD, FMN, and tetrahydrobiopterin. Generally, the three isoforms of NO synthase (NOS) have been labeled type 1 (nNOS), the neuronal isoform; type 2 (iNOS), the inducible isoform; and type 3 (eNOS), the endothelial isoform. NNOS and eNOS are constitutively expressed in the cells in which they are found. iNOS is not constitutively expressed, but rather is induced by a number of cytokines and lypopolysaccarides (LPS). It has been further discovered that nNOS serves as a neurotransmitter. iNOS, further, concerns host defense and cellular immunity. Also, vascular tone and hemodynamic control has been linked to eNOS. The three (3) isoforms of the NOS enzyme fall in the category of true isozymes since they share approximately 60% sequence homology.
iNOS has been specifically implicated in certain pathological diseased states. An article entitled xe2x80x9cExpression and Preferential Inhibition of Inducible Nitric Oxide Synthase in Aortas of Endotoxemic Ratsxe2x80x9d, by Weigert et al., Journal of the American Society of Nephrology, Volume 5, No. 12 (1995) discusses the functional importance of iNOS with respect to septic shock. Specifically, where sepsis and septic shock occurs, numerous cytokines and LPS from gram negative bacteria potentially can induce the expression of iNOS in monocytes, macrophages, neutrophils, hepatocytes, or other cell types, which leads to the overproduction of NO. This in turn leads to the deleterious effects associated with sepsis and septic shock due to extensive systemic vasodilation.
Various groups of researchers have reported on the development of monoclonal antibodies to NOS and on the utilization of such antibodies for biomedical experimentation. An article entitled xe2x80x9cStabilization of Inducible Nitric Oxide Synthase by Monoclonal Antibodiesxe2x80x9d by Hattori et al., Hybridoma, Volume 12, No. 6 (1993) states that a panel of monoclonal antibodies to rat iNOS was derived from activated rat peritoneal macrophages. It was reported therein that none of the monoclonal antibodies neutralized the enzymatic activity of rat iNOS, but some of the monoclonal antibodies stabilized the enzyme.
An article entitled xe2x80x9cTransient Expression of Calcium-Independent Nitric Oxide Synthase in Blood Vessels During Brain Developmentxe2x80x9d by Galea et al., FASEB Journal, Volume 9, (December 1995), describes a protein band which was detected with a monoclonal antibody raised against rat iNOS. Moreover, the Rengasamy article, prior identified, describes the development and characterization of a monoclonal antibody developed to bovine nNOS. Through western immunoblots, this monoclonal antibody was found to recognize bovine nNOS, bovine eNOS, and mouse iNOS. The same monoclonal antibody was found to recognize rat nNOS, rat eNOS, and rat iNOS, by immunohistochemical techniques.
An article entitled xe2x80x9cInducible Nitric Oxide Synthase In A Human Glioblastoma Cell Linexe2x80x9d by Fujisawa et al., Journal of Neurochemistry, Vol. 64 (1995) describes iNOS induction in A-172 cells, which is a human glioblastoma cell line.
An article entitled xe2x80x9cImmunochemical Detection of Inducible NO Synthase in Human Lungxe2x80x9d by Tracey et al., American Physiological Society, Rapid Communication (1994) describes iNOS induction in RAW 264.7 macrophages. Polyclonal antibodies raised against mouse iNOS derived from induced RAW 264.7 cells and were used to investigate the expression of iNOS in human lung tissue.
An article entitled xe2x80x9cCharacterization and Localization of Endothelial Nitric Oxide Synthase Using Specific Monoclonal Antibodiesxe2x80x9d by Pollock et al., American Physiological Society (1993) describes the development and characterization of a panel of monoclonal antibodies developed to bovine eNOS, which do not cross react with either nNOS or iNOS.
U.S. Pat. Nos. 4,376,110 and 4,879,219 describe immunoassays utilizing monoclonal antibodies to detect antigenic substances.
A brochure from Transduction Laboratories, Lexington, Ky., offers a number of mouse monoclonal antibodies raised to recombinant fragments of various rat isoforms of NOS.
A company called Santa Cruz Biotechnology in a brochure entitled xe2x80x9cSignaling Intermediatesxe2x80x94NOSxe2x80x9d offers a number of polyclonal anti-peptide antibodies specific for the various isoforms of NOS.
A brochure entitled xe2x80x9cIsostripxe2x80x9d by Boehringer Mannheim Corporation illustrates a simplified mouse monoclonal antibody isotyping kit which uses treated strips to detect mouse immunoglobulin subclasses, and kappa or lambda light chains.
The development of a panel of monoclonal antibodies to human iNOS for immunoassays specific for human iNOS would be a notable advance in the bio-medical field.
In accordance with the present invention a novel and useful panel of monoclonal antibodies specific to human iNOS have been developed and have been demonstrated to be useful in immunoassays that are specific for human iNOS.
The monoclonal antibodies have been characterized by a number of different standard techniques.
An object of the present invention is the development of immunoassays which can be used as clinical tests for hiNOS utilizing monoclonal antibodies specific to hiNOS.
Another object of the present invention is to develop a separate panel of polyclonal rabbit anti-peptide antibodies, which are specific for the three (3) isoforms of hNOS.
Yet another object of the present invention is to produce peptide sequences which mimic regions of hiNOS, and that bind to the monoclonal antibodies of the present invention.
A further object of the present invention is to provide truncated peptide sequences which mimic regions of hiNOS and that bind to the monoclonal antibodies of the present invention.
Another object of the present invention is to provide homolog peptides from proteins other than human iNOS to test the specificity characteristics of the monoclonal antibodies.
Yet another object of the present invention is to characterize the panel of monoclonal antibodies of the present invention to ascertain their individual utility in various assays and procedures.
The invention possesses other objects and advantages especially as concerns particular characteristics and features thereof which will become apparent as the specification continues.